Microbiology & Immunology - Theses
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ItemObesity impairs virus-specific memory CD8+ T cell signalling and function( 2023-03)The main function of the immune system is to protect the host against invading pathogens. Immunological memory is formed after a primary infection and serves to provide protection in a fast and vigorous manner upon secondary encounter with the same pathogen. Under obesity conditions, however, protective immunity is impaired, constituting a significant risk factor for high incidence and severity of re-infections. Indeed, during the current COVID-19 pandemic, obesity has been recognized as a major risk factor for adverse clinical outcomes. Impaired immunity observed in obese individuals has been attributed to a dysfunction in CD8+ T cells, which are essential for the elimination and sterile clearance of viral infections. Yet, the underlying mechanisms of the immune-compromised status of obese individuals remain poorly understood. Using mouse models of diet-induced obesity and lymphocytic choriomeningitis virus infection, we showed increased morbidity and mortality of obese mice after re-infection, recapitulating the clinical situation in humans. Obese mice failed to mount protective immunity and showed a profound loss of virus-specific memory CD8+ T cells in the spleen and liver. This phenotype was associated with a compromised proliferation capacity and reduced ability to produce the effector cytokines IFNg and TNFa. Furthermore, our data revealed a severe decline specifically within hepatic tissue-resident memory T cell pool, which positively correlated with the body weight and contributed to the severe symptoms observed in obese mice. Additionally, we observed enhanced accumulation of IgA+, IL-10 producing, and PD-L1+ B cells in the liver of obese mice, and their absence was associated with normal numbers of hepatic tissue-resident memory T cells. Notably, genetic ablation of IL-10 production by B cells did not reconstitute the memory response, indicating that IgA+ B cells do not exert a suppressive effect towards the virus-specific CD8+ T cells via IL-10. Furthermore, our findings show that the impaired memory response in obese mice is due to T-cell intrinsic mechanisms driven by long-term exposure of virus-specific memory CD8+ T cells to the inflammatory environment, rather than by affecting their development and recruitment. Molecular analysis revealed transcriptional reprogramming of memory CD8+ T cells under obesity conditions, resulting in impaired T-cell receptor signaling. The obesity-inflicted changes in memory CD8+ T cells were highlighted by the impairment of Ca2+ influx upon CD8+ T cell stimulation in vitro. Collectively, our findings indicate that virus-specific memory CD8+ T cells exposed to an obese environment lose their ability to confer protection. The future intention of this project is to identify key molecules within the Ca2+ signalling pathway that could be therapeutically modulated to improve the ability of virus-specific memory CD8+ T cells to provide protection and to improve the immune response of obese individuals to re-infections.
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ItemTargeting the HBV pregenomic RNA and viral mRNAs using the CRISPR-Cas13b system and determining the impact of HBV splice variants Sp3 and Sp9 on wildtype HBV replication( 2023-10)Chronic hepatitis B infection, caused by the hepatitis B virus (HBV), affects over 296 million people globally and almost a million people die annually because of hepatitis B associated complications such as liver disease and hepatocellular carcinoma (HCC). Upon infection, the relaxed circular DNA genome is released into the nucleus where it is converted into the covalently closed circular DNA (cccDNA) minichromosome. This is then transcribed into the HBV pregenomic RNA (pgRNA), which is the HBV replication intermediate, and the viral mRNAs, which together with the pgRNA encode the HBV proteins essential for replication. The pgRNA may also be spliced to produce smaller RNA forms, some of which impact HBV replication. There is global consensus that new HBV treatments are urgently required that target multiple stages of the HBV replication cycle to increase rates of functional HBV cure, defined as loss of HBV surface antigen, which is rarely achieved using current HBV therapies. The HBV pgRNA and viral mRNAs represent a novel target for new HBV treatments. Here, the bacterial CRISPR-Cas13b system, which targets RNA, was repurposed to target the HBV pgRNA, spliced RNAs and viral mRNAs to reduce HBV replication and protein expression, in pursuit of developing a potential new therapy for chronic HBV infection. This was the first study to repurpose CRISPR-Cas13b to target the HBV RNAs. CRISPR-Cas13b was first optimised to target the HBV RNAs using an in vitro transfection system. The efficacy of CRISPR-Cas13b targeting HBV was then tested using additional in vitro models and in an in vivo HBV model. A major barrier to developing more HBV treatments is that the HBV replication cycle and host/viral factors that influence HBV replication are not completely understood. One such aspect is the role of HBV splice variants, which are shorter DNA genomes, produced after reverse transcription of shorter RNA forms derived by splicing of the HBV pgRNA. The role of HBV splice variants in HBV replication and pathogenesis remains largely unknown, however an increased proportion of splice variants in patient sera has been associated with the development of liver disease and HCC. In addition, different HBV splice variants and their encoded novel fusion proteins have different effects on wildtype HBV replication in vitro. Here, the impact of two common but poorly characterised HBV splice variants, Sp3 and Sp9, on wildtype HBV replication and the impact of HBV splice variants on the host cell kinome was explored in vitro, to further investigate the role of HBV splice variants in HBV replication and pathogenesis, which may provide additional targets for new novel treatments.
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ItemDissecting the role of gd T cells in T cell priming for liver stage immunity( 2023-11)Liver resident memory T cells (TRM) are poised for protection against repeat infection and rapidly form a robust defence against tissue-specific insults such as liver stage malaria. A direct correlation between liver stage immunity and gd T cells has been observed both in mice (Zaidi et al. 2017) and in humans (Seder et al. 2013; Ishizuka et al. 2016), but the precise molecular mechanisms by which these gd T cells exert their protective effect are yet to be defined. In mice, intravenous injection with radiation-attenuated sporozoites (RAS) confers sterile protection against challenge with live sporozoites. This protection is mediated by responding antigen-specific CD8+ and CD4+ T cells that migrate to the liver and form resident-memory T cells (TRM). In the absence of gd T cells, protective CD8+ liver TRM are not generated, leaving mice susceptible to reinfection. Using Plasmodium-specific T cells as a readout for effective immunity, we determined that IL-4 is important for the accumulation of CD8+ and CD4+ T cells. By utilising complex in vivo systems including mixed-bone marrow chimeras and adoptive transfer of gd T cells, we revealed that gd T cell-derived IL-4 is crucial for the expansion of antigen-specific CD8+ T cells. In addition, in vivo neutralisation of IL-12 or IFN-g confirmed a partial dependency for these cytokines, despite their traditionally opposing function to IL-4. Given IL-4, IFN-g and IL-12 all have a clear role in CD8+ T cell priming following RAS vaccination, we hypothesised that IL-4 and IFN-g synergise to enhance cDC1 activity. These findings led to our development of a novel model to reconstitute cDC1-deficient mice using CRISPR-edited primary dendritic cells. This model enabled the investigation of the mechanism by which gd T cell derived IL-4 leads to DC activation and therefore effective CD8+ T cell expansion for memory development. Collectively, this project has shown a significant role for IL-4 in the priming of malaria-specific CD8+ T cells and demonstrates a novel pathway for collaboration between gd T cells, cDC1s, and CD8+ T cells, revealing the potential for harnessing gd T cells in vaccination strategies against malaria.
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ItemAntibody-mediated passive immunity against Helicobacter pylori(University of Melbourne, 2008)
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ItemThe role of T cells in the immunopathology of Helicobacter pylori infections in mice(University of Melbourne, 2007)
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ItemThe role of dendritic cells in CD8+T cell priming after epidermal HSV-1 infection(University of Melbourne, 2006)
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ItemInduction of immune responses by lipopeptide vaccines(University of Melbourne, 2006)
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ItemControl of pulmonary immunity by physical exercise( 2023-03)Sedentary lifestyles combined with high caloric nutrition are widely known to severely contribute to the rise of metabolic diseases like diabetes, atherosclerosis, or obesity in recent years. While endurance training has been shown to induce the secretion of adipokines, and so-called myokines (muscle-derived cytokines), the impact of physical exercise on the host immune response in the context of bacterial or viral infections remains largely unknown. Here, we aimed to investigate the impact of voluntary wheel running (VWR), mimicking an active lifestyle, on the pulmonary immune system and to which extent a lack of exercise might affect the severity of pneumonia induced by bacterial L. longbeachae or viral influenza A virus (IAV) infection. We observed that VWR enhanced stamina to exercise and reduced visceral adipose tissue. Moreover, VWR induced the expression of myokines and lipolysis-associated genes and decreased the number of circulating monocytes. Notably, neither acute nor long-term (2 and 8 weeks, respectively) physical exercise significantly affected the abundance or metabolism of pulmonary immune cells in healthy mice. However, upon infection with L. longbeachae, acute physical training reduced pathogen burden, dampened anorexia-induced weight loss, and decreased the recruitment of neutrophils and monocytes to the airways. Additionally, pro-inflammatory cytokines associated with bacterial clearance, including IFN-g & TNFa increased in the lungs of exercised mice. Notably, VWR enhanced the potential to produce TNFa in both alveolar macrophages and infiltrating monocytes early and late in infection with L. longbeachae. Furthermore, in running mice we found increased mitochondrial and glucose dependency in myeloid cells, crucial for the inhibition of pathogen replication. Our results also show that CD4+ T cells from trained animals display reduced IFN-g production, suggesting that exercise may predominantly boost the innate immune response. In contrast, during IAV infection, we observed significantly decreased numbers of activated type 1 helper T (Th1) cells in running mice, critical for viral clearance. However, the frequency of tetramer+ CD4+ and CD8+ T cells was increased, indicating higher antigen specificity of the immune response. Additionally, we found higher viral RNA content in lung tissue from exercising mice, and elevated production of pro-inflammatory cytokines including type I interferons. Moreover, we found higher expression of interferon-signalling genes in the pulmonary tissue of exercising mice. VWR increased the gene expression of Ifng in CD44+ CD4+ T cells in running mice, suggesting an enhanced capacity of CD4+ T cells to produce IFN-g. Notably, VWR increased the expression of tissue-residency markers on CD8+ T cells. Taken together, our results suggest that VWR might have opposing effects on pulmonary immunity during infection. Hence, we conclude that acute physical exercise might enhance protection against bacterial invasion (L. longbeachae) by specifically boosting the innate immune response. In contrast, VWR reduces Th1-mediated anti-viral responses and increases the pulmonary viral RNA content, suggesting that physical exercise might enhance the susceptibility to IAV infection. However, by boosting the adaptive IFN-g-mediated anti-viral response, antigen-specificity, and increasing tissue residency, exercise may enhance the defence against secondary viral infections.
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ItemThe Development, Homeostasis, and Function of Unconventional T Cells( 2023-09)Unconventional T cells detect non-peptide antigens presented by MHC class-I-like molecules, such as MR1 and CD1. MR1-reactive T cells, CD1-reactive T cells, and gamma-delta T cells represent three broad unconventional T-cell lineages which collectively are abundant and play key roles in the immune response. The frequencies of these cells vary widely between individuals, and the factors that govern their numbers and diverse effector functions are not well-understood. This thesis investigates the mechanisms that controls the development, homeostasis, and function of unconventional T cells and their subsets in the thymus and peripheral tissues. In chapter 3, MR1 and group 1 CD1 (CD1a, CD1b, and CD1c) tetramers were used to isolate and characterise rare unconventional T-cell populations in the human thymus. Using tetramer-mediated enrichment, thymic MR1-reactive T cells were found to comprise Valpha7.2+ and diverse Valpha7.2- subsets that differed in phenotype, binding to antigen-loaded MR1 tetramers, TCR repertoire diversity, frequency, and post-natal expansion. Whilst tetramer-mediated enrichment allowed for isolation of thymic CD1a- and CD1b-reactive T cells, blockade of CD36 expressed on thymocytes was additionally needed in order to detect CD1c-reactive T cells. Group 1 CD1-reactive T cells were highly rare after enrichment and generally resembled other CD4+ thymocytes. These findings highlight diverse MR1 and CD1-reactive T cell subsets in the thymus and forms the basis for understanding their intrathymic generation and developmental pathways. The expansion of MAIT cells in NKT- and gamma-delta T cell-deficient mice was investigated in chapter 4. Although MAIT cells were highly elevated in mice deficient in both NKT and gamma-delta cells, they phenotypically and functionally resembled their counterparts in wildtype mice. Mechanistically, this MAIT cell expansion was due to: 1) increased rearrangement of the MAIT cell TCR alpha chain within developing Tcrd-/- thymocytes, and 2) a higher capacity of peripheral MAIT cells to proliferate in the absence of NKT and gamma-delta cells. Overall, this chapter provides evidence for a shared niche in which MAIT, NKT, and gamma-delta T cells reside and compete for common homeostatic factors, revealing a novel interplay between their steady-state frequencies. In chapter 5, the regulation of peripheral unconventional T cells by the purinergic P2RX7 receptor was examined. Human unconventional T cells expressed P2RX7, whereas their mouse T-bet+, but not RORgt+, counterparts highly co-expressed the ADP-ribosyltransferase, ARTC2, and P2RX7. P2RX7 activation in response to ATP induced death of both mouse and human unconventional T cells ex vivo. Mouse T-bet+ unconventional T cells were highly susceptible to the effects of ARTC2-dependent P2RX7 activation in response to nicotinamide adenine dinucleotide, which resulted in their cell death ex vivo and depletion in vivo. By blocking ARTC2 or P2RX7, this chapter demonstrated the existence of IFN-gamma/IL-4 co-producing unconventional T cells, including MAIT and other non-MAIT/NKT alpha-beta T cell populations, which were selectively regulated by ARTC2-dependent P2RX7 activation. In contrast, this axis did not affect IL-17-producing unconventional T-cell subsets. These findings reveal a unique mechanism that controls the number and functional diversity of unconventional T cells via the selective regulation of their T-bet+ and IFN-gamma/IL-4 co-producing subsets. Overall, this thesis has examined the presence of diverse unconventional T cells in the human thymus, their regulation within a homeostatic niche, and their modulation by P2RX7 activation. Whilst unconventional T cells are collectively abundant, understanding their development and homeostasis will provide insight into why their frequencies vary widely in humans and how their numbers can be finely-controlled. The homeostatic relationships between unconventional T cells and how their functional subsets are regulated will be important considerations in the targeting of one or more unconventional T-cell populations within the immune response. Given their production of functionally-opposing cytokines, findings in this thesis will guide the development of future immunotherapies that leverage the abundance and potent effector functions of unconventional T cells in treating disease.
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ItemA multi-omic approach to understanding the mechanisms of daptomycin resistance in Enterococcus faecium( 2023-11)Healthcare-associated infections caused by multi-drug resistant organisms such as vancomycin-resistant Enterococcus faecium (VREfm) are a public health threat. Daptomycin is a ‘last-resort’ antibiotic for VREfm infections with a novel membrane targeting mode-of-action, but for which resistance has surprisingly been reported in clinical strains. Despite the importance of daptomycin for treating VREfm infections, the genetic changes and molecular mechanisms leading to daptomycin resistance are poorly characterised in VREfm. This thesis aims to better understand these mechanisms using a ‘multi-omic’ approach – combining genomics, transcriptomics, proteomics, and lipidomics – on defined isogenic mutants and clinical VREfm strains. To understand which genetic mutations were associated with daptomycin resistance in clinical VREfm, we completed a genomic epidemiological and phenotypic study in Chapter 2. We demonstrate that daptomycin resistance is linked with the presence of novel mutations (G482D, H486Y, and S491F) in the B subunit of the bacterial RNA polymerase (RpoB) that confer resistance to rifaximin and cross-resistance to daptomycin. Surprisingly, these RpoB mutations emerge in VREfm following exposure to rifaximin, an unrelated antibiotic that is commonly prescribed to liver disease patients, a cohort at high-risk for VREfm colonisation and infection. Clinical VREfm isolates with these RpoB mutations were spread globally, across 20 countries and 5 continents, making this a major mechanism of resistance. Our study shows that rifaximin use may be compromising the clinical use of daptomycin through the selection RpoB mutations in VREfm. To understand how mutations in the B subunit of the bacterial RNA polymerase cause resistance to daptomycin, a cell membrane active antibiotic, we utilised a multi-omics approach in Chapter 2. Our analyses show that the G482D, H486Y, and S491F RpoB mutations mediate daptomycin resistance via a conserved mechanism, with each of these RpoB mutations causing similar transcriptional reprograming in VREfm. The dysregulation of a single, previously uncharacterised genetic locus was found to be solely responsible for daptomycin resistance. The locus, that we have named the Phenotypic Resistance to Daptomycin (prd) operon consists of a transcriptional regulator (PrdR) and two putative membrane proteins (PrdA and PrdB). Upregulation of the prdRAB operon leads to cell membrane remodelling, with decreased levels of anionic phospholipids (PG and CL) and increased levels of cationic phospholipids (Lys-PG). This ultimately decreases the negative cell surface charge and reduces daptomycin binding, which renders VREfm resistant to daptomycin. Mutations in the cell membrane stress response system LiaFSR are a major mediator of daptomycin resistance in VREfm, however, the molecular mechanism is unknown. We therefore created a panel of isogenic VREfm mutants and applied these same multi-omic approaches to understand how the clinically common mutations (namely LiaR W73C and LiaS T120A) in LiaFSR lead to changes in daptomycin susceptibility in Chapter 3. Our analyses show that mutations in LiaRS result in the dysregulation of several effector proteins, such as PrdRAB, LiaX, and a HD domain protein, all associated with daptomycin resistance. The LiaRS mutants displayed changes in cell membrane remodelling (decreased PG and CL and increased Lys-PG) and decreases in negative cell surface charge, leading to daptomycin resistance through reduced binding of the antibiotic. Through this work, we have identified a previously uncharacterised, but globally disseminated, daptomycin resistance mechanism mediated through mutations in the rpoB gene. Further, by understanding the molecular mechanism of daptomycin resistance in two major systems, RpoB and LiaRS, we demonstrate that although these mutations (RpoB G482D, H486Y, and S491F as well as LiaR W73C and LiaS T120A) occur in unrelated genes, they cause upregulation of the same operon (PrdRAB), leading to similar phenotypic changes to the cell membrane. These data indicate that electrostatic repulsion is an important mechanism of daptomycin resistance in VREfm.